Biological activity, UHPLC-MS phytochemical profiling, and computational studies of the leaf extract of Acridocarpus socotranus

Abstract

Soqotra Island is renowned for its exceptional biodiversity, harboring approximately 825 plant species, 307 of which (37%) are endemic. Despite its rich traditional knowledge, many of these plants remain scientifically underexplored. This study, for the first time, investigates the phytochemical composition, biological activities, and molecular interactions of Acridocarpus socotranus, an endemic species traditionally used to treat rheumatism and skin disorders. A comprehensive UHPLC-MS analysis of the methanol leaf extract identified 25 phytochemicals, classified into seven major categories: alkaloids, terpenoids, flavonoids, coumarins, glycosides, steroids, and other organic compounds. The extract exhibited a high phenolic content (95.6 ± 4.90 mg GAE/g). Enzyme inhibition assays revealed that the extract effectively suppressed the activity of several enzymes, including mushroom monophenolase (IC₅₀: 125 µg/mL), diphenolase (IC₅₀: 191.07 µg/mL), xanthine oxidase (IC₅₀: 127.8 µg/mL), and glyoxalase I (IC₅₀: 0.27 µg/mL). Cytotoxicity testing showed that the extract was very effective at stopping the growth of colorectal cancer (SW480 and HCT116) cells (IC₅₀: 259.7 ± 12.1 and 146.3 ± 10.9 µg/mL, respectively), but it was less effective against breast cancer (MCF7 and MDA-MB-231) and prostate (PC3) cell lines (IC₅₀ > 350 µg/mL). . Molecular docking further confirmed these findings, revealing that phytochemicals from A. socotranus can effectively bind to glyoxalase I and to a conserved pocket of the salivary protein tablysin-15. The observed binding patterns resemble those of known antagonists that neutralize inflammatory mediators rather than blocking their receptors. These antagonist-like interactions suggest a potential anti-inflammatory mechanism, thereby providing additional therapeutic value.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-29208-7.

Introduction

Enzymes are involved in various pathological conditions, including diabetes, microbial infections, HIV, neoplastic diseases, inflammation, and skin disorders. Inhibiting these enzymes is considered a viable strategy for treating such conditions and for elucidating their underlying mechanisms. The development of effective enzyme inhibitors represents a critical step in the drug discovery and development process^1,2.

Melanin is essential for protecting human skin against ultraviolet radiation; however, excessive melanin accumulation can result in pigmentation disorders such as senile lentigines, freckles, melasma, and ephelides³. Tyrosinase (TYR), also referred to as polyphenol oxidase, is a key enzyme that regulates melanin synthesis in melanocytes, which reside in the basal layer of the epidermis^4,5. Over the past decade, numerous moderate to potent tyrosinase inhibitors derived from both synthetic and natural sources have been identified⁶. Nevertheless, many of these compounds have exhibited limitations, including toxicity or reduced efficacy in clinical evaluations^7,8.

Xanthine oxidase (XO) plays a central role in purine metabolism by catalyzing the oxidation of hypoxanthine to xanthine, followed by the conversion of xanthine to uric acid. Inhibition of XO is therefore a key strategy for reducing uric acid levels. Several XO inhibitors, including oxypurinol, allopurinol, and febuxostat, are widely used in the clinical management of gout and hyperuricemia. Despite their efficacy, these drugs are associated with notable adverse effects, such as allergic reactions, nephritis, and hepatitis, which limit their long-term use⁹.

Glyoxalase I (Glo-I) is a key enzyme responsible for detoxifying methylglyoxal (MG) into D-lactate, using glutathione as a cofactor. MG, a highly reactive byproduct of glycolysis, can react with proteins and DNA to form stable adducts. Inhibition of Glo-I, whether by radiation or medications, results in the accumulation of MG in tumor cells, thereby inducing apoptosis^10,11. These observations highlight the urgent necessity for novel inhibitors of TYR, XO, and Glo-I that exhibit high efficacy and minimal adverse effects.

Soqotra is of global significance for biodiversity conservation due to its exceptionally rich and unique flora. Botanists estimate that the island is home to hundreds of plant families. Among the more than 800 plant species recorded, 307 are considered endemic¹². The genus Acridocarpus possesses a variety of phytochemicals, including triterpenes such as beta-sitosterol, stigmasterol, friedelin, oleanolic acid, and ursolic acid, as well as flavonoids including morin, morin-3-O-β-D-glucopyranoside, apigenin, luteolin, vitexin, kaempferol, and quercetin 13–¹⁵. Traditionally, certain Acridocarpus species have been used in folk medicine to treat malaria, pain, infections, blood disorders, infertility, gastrointestinal issues, paralysis, and skin blisters (pemphigus)^16,17.

Acridocarpus socotranus Oliv. (Malpighiaceae), endemic to Soqotra and locally known as “Kerella,” is primarily found in the Hajar Mountains. Its dried leaves have traditionally been used to heal wounds and alleviate rheumatism¹⁸ According to Mothana et al., A. socotranus exhibits antibacterial and antioxidant activities, likely attributable to its flavonoid and terpenoid content¹⁹. A. socotranus was selected for this study based on its traditional use in treating rheumatism and skin disorders. This study presents the first in-depth characterization of the phenolic composition and UHPLC–MS phytochemical profile of A. socotranus, coupled with a systematic evaluation of its inhibitory activities against tyrosinase, glyoxalase, and xanthine oxidase, as well as its cytotoxic potential. Furthermore, advanced computational approaches, including molecular docking and molecular dynamics simulations, were employed to elucidate the molecular mechanisms underlying its observed bioactivities.

Methodology and material

Plant materials and extraction

The plant material was collected from Soqotra Island during March–April 2021. Taxonomic identification was performed by botanist Dr. Abdulwali Alkhulaidi, and voucher specimens (ASO2301) were deposited in the Pharmacognosy Department, Faculty of Pharmacy, Sana’a University, Yemen. Fifty grams of air-dried, powdered plant material were extracted in a Soxhlet apparatus with 500 mL of methanol under reflux for 8 h. The resulting extract was filtered and concentrated to dryness under reduced pressure at 40 °C using a rotary evaporator, yielding 8 g (16%). The dried extract was stored at − 20 °C until further analysis.

Phytochemical composition

Total phenolic content

The total polyphenolic content (TPPC) of the plant extract was determined using the Folin–Ciocalteu method, following the procedure described by Chen et al. with little modifications²⁰ and the results were expressed as milligrams of gallic acid equivalents per 100 mg of dried extract (mg GAE/100 mg)²⁰. Further details are provided in the Supplementary Text.

UHPLC-MS Secondary Metabolites Profiling.

UHPL C Accurate -Mass Q -TO F (Agilent 1290 Infinity LC system coupled to Agilent 6520) mass spectrometer with dual ES I source was used, as described in previous literature²¹. The details are presented in the Supplementary TABLE S1 and S2.

Biological activities

Enzyme inhibition assays

Mushroom tyrosinase (25 kU), kojic acid, L-tyrosine, and L-DOPA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was obtained from Tedia (USA). Enzyme activity was measured using a Cytation 5 BioTek^® microplate reader (Agilent, USA) equipped with Gen5 software. The dried extract was dissolved in DMSO to form a stock solution of 20 mg/ml and then diluted with either 50mM phosphate buffer, pH 6.5, or DMEM solution to a concentration of 1 mg/ml (5% DMSO). The concentrations of proper dilutions were prepared and used in the assays, where the concentration of DMSO did not exceed 1%. The Enzyme inhibition assays against the following therapeutically important enzymes, mushroom monophenolase and diphenolase tyrosinase (TYR), glyoxalase (Glo-I), and xanthine oxidase (OX) were evaluated using previously described protocols^7,22–24. Positive controls were used as Kojic acid and arbutin for tyrosinase inhibitory activity, allopurinol for xanthine oxidase inhibitory activity, and myricetin for the glyoxalase inhibitory activity. The details are presented in the Supplementary Text.

Cytotoxic activity

Human colorectal cancer cell line (HCT116 and SW480) and human breast cancer cell lines (MCF7 and MDA-231), Prostate cancer (PC3) cell lines, and normal skin fibroblast were purchased from ATCC (USA). Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco, USA) was used to propagate HCT116, SW480, MDA-231, and normal skin fibroblast cell lines, while RPMI culture media augmented with 10% FBS was used to grow MCF7 and PC3 cells. The cells were incubated at 37 °C in 5% CO2. A stock solution of plant dried extract in DMSO (50 mg/ml) was prepared, and different dilutions were prepared in culture media. An appropriate number of each trypsinized cell line was transferred to each well and incubated for 24 h. Then, the cells were treated in triplicate by the different concentrations of the plant extract and incubated for another 72 h. The cellular survival was evaluated by the reduction of 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) to the colored formazan, which was spectrophotometrically measured at 570 nm^24,25.

Computational studies

Protein model preparation.

The co-crystallized structures of the human GLO-I enzyme were prepared using Discovery Studio 2021, with 3D coordinates retrieved from the Protein Data Bank (PDB ID: 3VW9)²⁶. The selected structure, with a resolution of 1.47 Å, represents the human GLO-I enzyme complexed with an N-hydroxypyridone inhibitor (HPJ)²⁶. To determine the other protein used crystal structure of the tablysin-15-leukotriene E4 complex (PDB ID: 3U3U)²⁷, to prepare the retrieved co-crystallized structures for docking analysis, the Molecular Operating Environment (MOE) software (Inc, 2016) was used. The preparation process included the removal of water molecules and associated cofactors, the addition of hydrogen atoms, and the reconstruction of any missing atoms. Energy minimization was performed using the AMBER10: EHT force field, applying a 0.01 rms kcal/mol gradient to resolve steric clashes and optimize bond parameters. Additionally, the structural topology of the protein was carefully examined to identify and address any irregularities.

Molecular Docking

Docking simulations were performed using the MOE-Dock module within the MOE 2019 software (Chemical Computing Group, Montreal, Canada). The binding site was defined based on the co-crystallized inhibitor present in the reference crystal structure, and further validated using the Site Finder tool. Dummy atoms were created at the designated residue locations to specify the docking site. Unlike grid-based docking programs, MOE does not require explicit grid box dimensions; instead, docking is confined to the active site cavity defined by these dummy atoms.

The ligand database consisted of phytoconstituents identified through UHPLC-MS, along with standard inhibitors relevant to the proteins being studied. Ligands were protonated at physiological pH (7.4), energy minimized, and subsequently docked into the defined active sites. The docking process employed the Triangle Matcher algorithm for ligand placement, London dG scoring for ranking, and rigid receptor refinement. For each ligand, 100 poses were generated, and the top-scoring pose was selected for further evaluation. The most favorable protein–ligand conformations were analyzed based on docking scores and their interactions with key active site residues.

Statistical analysis

The final results were expressed as mean ± SD. All samples were tested in triplicate, and the group means were compared using an ANOVA test.

Results and discussion

Phytochemical composition

Phenolic content

Phenolic compounds constitute key plant components possessing redox attributes that contribute to antioxidant activity and provide multiple protective and therapeutic properties against various conditions, including bacterial, protozoal, fungal, and viral infections, as well as inflammation, diabetes, cancer, and aging problems. Phenolics are among the phytochemicals that may assist in finding leads or novel therapeutic drugs²⁸. The dried extract yield in this study was higher than the 10.5% reported for A. socotranus¹⁹ but lower than those of A. orientalis and A. monodii leaf extracts^16,29. The total phenolic content of A. socotranus was measured using the Folin–Ciocalteu method and is shown in Table 1 as gallic acid equivalent, based on the standard curve (Y = 0.0102X + 0.0222; R2 = 0.9979). The total phenolic content of A. socotranus (95.6 ± 4.90 mg GAE/g) was lower than that reported for A. orientalis leaves from Oman and the UAE (149.23 ± 2.84 and 184.24 ± 4.39 mg GAE/g, respectively) and A. monodii leaves from Mali (109.82 ± 2.36 mg GAE/g)^16,30. This variation in the phenolic contents may be due to geographical locations, environmental factors such as soil composition, temperature, rainfall, and ultraviolet radiation incidence³¹, or factors including extract volume, reagent selection, reaction time, color development temperature, measurement wavelength, and reference substance may affect the TPC values³².

Table 1.

Yield, polyphenolic content and biological activities of A. socotranus extract.

Plant species (Family)	Yield	Polyphenolic content	Monophenolase inhibition (Tyrosine)	Diphenolase inhibition (L-dopa)	(Glo I) Glyoxalase inhibition	XO inhibition
	%	mgGAE/100 mg	IC50 (µg/ml)	IC50 (µg/ml)	IC50 (µg/ml)	IC50 (µg/ml)
A. socotranus extract	16%	55.7 ± 4.9	125	191.07	0.27	127.8
Allopurinol						90.2 ± 1.94(this is %inhibition, IC50 is much less)
Kojic acid			9.37	42.5

UHPLC–MS analysis

The biological potential of medicinal plants is largely attributed to the bioactive constituents naturally present in their extracts. To accurately assess the biological effects of plant extracts, secondary metabolite elucidation is crucial³³. Consequently, to obtain a comprehensive characterization of the various phytochemicals, UHPLC–MS analysis of the methanol leaf extract of A. socotranus was conducted in negative ionization mode. LC/MS analysis of A. socotranus revealed 25 compounds for the first time, classified into eight phytochemical groups: alkaloids, flavonoids, terpenoids, glycosides, steroids, coumarins, organic acids, and other compounds (Table 2; Fig. 1 and Supplementary compounds chromatograms).

Table 2.

The cytotoxic activity of A. socotranus (represented by its IC50’s) against different cancer cell lines.

Cell line	Acridocarpus (IC50 (µg/ml)	Doxorubicin (µM)
HCT116	259.7 ± 12.1	1.0 ± 0.12
SW480	146.3 ± 10.9	60.6 ± 4.8
MDA-MB-231	357.6 ± 17.4	1.3 ± 0.1
MCF7	R	5.1 ± 0.9
PC3	378.2 ± 16.3	2.5 ± 0.7
Normal skin fibroblast	107.7 ± 9.4	0.044 ± 0.03

Fig. 1.

Active site’s pocket by the co-crystallized ligands in the 3VW9 crystal.

Alkaloids were reported for the second time in the Acridocarpus genus16 (Konaré et al., 2024), whereas other phytochemical groups, such as terpenes, have previously been isolated and characterized in A. vivy leaves.

Alkaloids were reported for the second time in the Acridocarpus genus¹⁶, while other phytochemicals have been previously reported in leaves of A. vivy (terpenes)¹⁵, A. chloropterus, and A. orientalis (flavonoids)^13,14. Nigrifactin alkaloid is reported for the first time in A. socotranus, having previously been isolated only from Streptomyces species³⁴. Nigrifactin and poisonous coniine from Conium maculatum are both piperidine alkaloids, sharing a similar core chemical structure. The structural similarity may explain the neurotoxic effects of A. socotranus leaves on camel skeletal muscles, as reported by local informants following substantial grazing^12,18.

Biological activity

Enzyme inhibition potential.

The enzyme inhibitory activities are summarized in Table 1. The extract showed the strongest inhibitory activity against glyoxalase (IC50 0.27 µg/ml), followed by monophenolase (IC50 125 µg/ml), diphenolase (IC50 191.07 µg/ml), and xanthine oxidase (IC50 127.8 µg/ml). The enzyme inhibitory activities may be attributed to its bioactive constituents (phenolics and flavonoids), as reported by several researchers in previous studies^10,35–37. The extract showed the strongest inhibitory activity against GLOI with IC₅₀ of 0.27 µg/ml compared to myricetin, correlating with cytotoxic effect against human colorectal cancer cell lines (HCT116 and SW480), exhibiting an IC50’s of 259.7 ± 12.1 and 146.3 ± 10.9 µg/ml, respectively, as GLOIs demonstrate prospective as therapeutic options in colorectal cancer (CRC) by decreasing cancer growth, migration, and invasion³⁸.

Cytotoxicity.

The methanol extract of A. socotranus showed a significant cytotoxic activity against colorectal cancer cells (SW480 and HCT116) with an IC50 of 146.3 ± 10.9 and 259.7 ± 12.1 (µg/ml), respectively, and low activity against human breast cancer cells (MDA-MB-231 and MCF7) and prostate cancer (PC3) with IC50 > 350 (µg/ml) (Table 2). Derivatives of fluoren-9-one have previously been reported to inhibit SNU398 hepatocellular carcinoma cells³⁹. Although the plant’s activity against breast cancer cells is low, two compounds identified in the plant, 11-hydroxytephrosin and hinokitiol, possess marked cytotoxic activity against human breast cancer cells (MCF7) according to previous studies^40,41. Moreover, hinokitiol displayed higher inhibition in colon cancer cells, HCT-116 and SW480, compared to normal colon cells⁴². Other species of the Acridocarpus genus have also been shown to possess anticancer properties. For instance, free morin and its glycosides were identified as the active compounds responsible for the inhibition of breast cancer cell proliferation in A. orientalis leaf extract^13,43,44. Acridocarpusic acid C, isolated from A. vivy exhibited significant cytotoxic activity against the A2780 human ovarian cancer cell line (performed at Virginia Polytechnic Institute and State University), with an IC₅₀ value of 0.7 µg/mL¹⁵. Furthermore, (2 S,4R)−4-(9 H-pyrido[3,4-b]indol-1-yl)−1,2,4-butanetriol has been proposed as a potential therapeutic agent for monkeypox⁴⁵. Chemically, it is classified as a harmala alkaloid due to its structural similarity to alkaloids found in Peganum harmala seeds, such as harmalicidine, harmine, peganine (vasicine), and vasicinone, all of which have demonstrated cytotoxic activity against cancer cell lines⁴⁶. Deterrol has also been reported to exhibit moderate cytotoxicity against ECA cells (Ehrlich ascitic tumor cells) and L1210 cells (mouse lymphocytic leukemia, ATCC CCL 163)⁴⁷.

Table 3.

UHPLC-MS analysis of Acridocapus Socotranus methanol extract (negative ionization mode).

S.No	Compound Class	RT	Mass m/z	Name	Mol. formula
0.1	Pyrrolizine alkaloids	0.616	201.1162	2,3-Dihydro-6-methyl-5-(5-methyl-2-furanyl)−1 H-pyrrolizine	C13 H15 N O
2	Fluorenes (Benzenoids)	0.639	180.0581	Fluoren-9-one	C13 H8 O
3	Indole alkaloids	0.644	272.1173	(2 S,4R)−4-(9 H-Pyrido[3,4-b]indol-1-yl)−1,2,4-butanetriol	C15 H16 N2 O3
4	Amino acid (methionine derivatives) Peptide	0.645	248.0834	N-Formylmethionylalanine	C9 H16 N2 O4 S
5	Monoterpenoid	0.661	326.1382	Hinokitiol glucoside	C16 H22 O7
6	Flavonoids (Flavanone)	0.662	324.1008	5,6-Dimethoxy-[2’’,3’’:7,8]furanoflavanone	C19 H16 O5
7	Flavonoids (Flavones)	0.662	358.086	5,7-Dihydroxyflavone 7-benzoate	C22 H14 O5
8	Isoflavonoids	0.68	252.0798	7-Hydroxy-2-methylisoflavone	C16 H12 O3
9	Cinnamaldehydes	0.684	192.0604	2-(Methylthiomethyl)−3-phenyl-2-propenal	C11 H12 O S
10	Gallic acid derivative	1.662	344.0736	Theogallin	C14 H16 O10
11	Leucoanthocyanidin	7.407	290.0782	Epifisetinidol-4alpha-ol	C15 H14 O6
12	Phenolic acid	7.408	138.0313	p-Salicylic acid	C7 H6 O3
13	Flavonoid glycoside (Flavones; flavanol)	7.587	546.1722	Flavonol 3-O-[alpha-L-rhamnosyl-(1–6)-beta-D-glucoside] = Flavonol 3-O-rutinoside	C27 H30 O12
14	Fatty acyl glycoside O-Acyl carbohydrate Monoterpene glycoside	7.84	386.1925	Corchoionol C 9-glucoside	C19 H30 O8
15	Flavonoid	7.848	436.1363	Ent-afzelechin-7-O-beta-D-glucopyranoside	C21 H24 O10
16	Glycoside Benzenoid glucoside	7.849	284.0895	D-Vacciniin	C13 H16 O7
17	Piperidine alkaloid	8.639	175.1363	Nigrifactin	C12 H17 N
18	Flavonoid	8.786	426.1332	11-Hydroxytephrosin	C23 H22 O8
19	Lactone glycoside	8.788	264.0832	3-Hydroxy-4-butanolide	C10 H16 O8
20	Flavonoid (Flavan-3-ols)	9.044	442.0895	Epicatechin Monogallate	C22 H18 O10
21	Nonanoic acid (organic acid)	9.193	174.1259	(+)−3-hydroxy pelargonic acid	C9 H18 O3
22	Fatty acid ester	15.824	212.1775	7Z-Undecenyl acetate	C13 H24 O2
23	Steroid	16.73	638.3653	26-Glucosyl-1,3,11,22-tetrahydroxyergosta-5,24-dien-26-oate	C34 H54 O11
24	Coumarin	16.746	126.0317	4-Hydroxy-6-methylpyran-2-one	C6 H6 O3
25	Sesquiterpenoid	19.742	478.3826	Deterrol stearate	C33 H50 O2

Computational study

Molecular docking

Structural similarity between natural compounds and standard inhibitors

Glyoxalase-I (GLO-I) is the primary enzyme in the glyoxalase system responsible for detoxifying cytotoxic α-ketoaldehydes like methylglyoxal. Due to its overexpression in several carcinomas, including breast, colorectal, prostate, and bladder cancer, GLO-I has been recognized as a promising therapeutic target in cancer treatment.

In this study, the methanol extract of A. socotranus demonstrated significant GLO-I inhibition, with an IC₅₀ value of 0.27 µg/mL, surpassing the standard inhibitor, myricetin (IC50 = 0.84 µg/mL). The inhibition mechanism was analyzed by comparing the phytoconstituents identified via UHPLC-MS with co-crystallized standard inhibitors, including 1-hydroxy-6-[1-(3-methoxypropyl)−1 H-pyrrolo[2,3-b]pyridin-5-yl]−4-phenylpyridin-2(1 H)-one (HPJ), 4-(2-hydroxyethyl)−1-piperazine ethane sulfonic acid, and zinc.

Figure 1 illustrates the active site occupancy of co-crystallized ligands. The N-hydroxypyridone ligand in the 3VW9 crystal selectively binds to the hydrophobic pocket and zinc region. Structurally, GLO-I is a homodimeric zinc metalloenzyme with an active site located at the interface of two chains, divided into three key regions: a deep hydrophobic pocket, a central zinc ion, and a positively charged entry site. The Zn²⁺ ion coordinates amino acid residues from both chains, playing a critical role in catalytic function.

Docking validation and phytoconstituent interactions.

To validate the docking process, HPJ was redocked into the GLO-I active site (PDB ID: 3VW9), yielding a binding energy of −14.74 kcal/mol. The binding energies of 23 phytoconstituents from A. socotranus ranged from − 10.16 to −5.02 kcal/mol, indicating strong binding affinity (Table 4). The N-hydroxypyridone derivative was found to coordinate with the zinc cation (d(NOZn) = 2.4 Å, d(COZn) = 2.2 Å), with its 4-phenyl group forming an edge-to-face interaction with Phe62A (4.3 Å) within the hydrophobic pocket.

Table 4.

Binding energy of phytoconstituent of methanol extract of A. socotranus.

	Compound class	compound	Binding energy 3VW9	Binding energy 3U3U
1	Pyrrolizine Alkaloids	2,3-Dihydro-6-methyl-5- (5-methyl-2-furanyl)−1 H- pyrrolizine	−5.02	−6.17
2, 3,4	Fluorenes (Benzenoids), Harmala alkaloid and methionine dervatives	Fluoren-9-one, (2 S,4R)−4-(9 H-Pyrido[3,4- b]indol-1-yl)−1,2,4- butanetriol N- Formylmethionylalanine	−5.74	−5.34
5	monoterpenoid.	Hinokitiol glucoside	−7.85	−7.08
6	Flavonoids	5,6-Dimethoxy- [2’’,3’’:7,8]furanoflavanone	−6.77	−5.54
7	flavonoids	5,7-Dihydroxyflavone 7- benzoate	−10.16	−6.81
8	Isoflavonoids	7-Hydroxy-2- methylisoflavone	−5.74	−6.74
9	cinnamaldehydes	2-(Methylthiomethyl)−3- phenyl-2-propenal	−8.37	−6.06
10	Gallic acid derivatives	Theogallin	−9.09	−5.40
11	leucoanthocyanidin	Epi fisetin idol-4alpha-ol	−7.45	−5.54
12	Phenolic acid	p-Salicylic acid	−6.84	−4.99
13	Flavonoids	Flavonol 3-O-[alpha-L- rhamnosyl-(1->6)-beta-D- glucoside]	−8.67	−7.23
14	Fatty acyl glycosides	Corchoionol C 9- glucoside	−6.65	−6.78
15	Flavonoid	Ent-afzelechin-7-O-beta- D-glucopyranoside	−7.65	−6.47
16	Glycosides	D-Vacciniin	−8.29	−6.68
17	Piperidine alkaloid	Nigrifactin	−5.95	−5.63
18	flavonoids	11-Hydroxytephrosin	−6.11	−5.30
19	Lactones	3-Hydroxy-4-butanolide	−9.15	−7.13
20	Flavonoids (flavan- 3 ols)	Epicatechin Monogallate	−6.73	−6.19
21	nonanoic acid organic acid	(+)−3-hydroxy pelargonic acid	−8.34	−6.25
22	Fatty acid ester	7Z-Undecenyl acetate	−6.44	−7.47
23	Steroid	26-Glucosyl-1,3,11,22- tetrahydroxyergosta-5,24- dien-26-oate	−7.74	−7.56
24	Coumarin	4-Hydroxy-6-methylpyran- 2-one	−5.02	−4.59
25	sesquiterpenoids	Deterrol stearate	−7.60	−9.18
	Crystal structure 1		−14.74	−9.89
	Crystal structure 2- for PROTEIN 3U3U			−14.72
	Crystal structure 1-for PROTEIN 3U3U			−15.15

Among the phytoconstituents, cinnamaldehyde exhibited the highest binding affinity (−10.16 kcal/mol), binding to zinc at the same site as HPJ, interacting with Glu99, Glu172, and Glu33 in the hydrophobic GSH binding pocket, and forming an edge-to-face interaction with Phe67A (Fig. 2). Additionally, fluorenes, harmala alkaloids, and methionine derivatives also exhibited strong zinc binding.

Fig. 2.

Interaction between ligands (compound 7; cinnamaldehyde) and compound 2 (PDB ID: 3VW9).

The zinc ion at the core of the GLO-I active site is essential for detoxifying methylglyoxal into lactic acid. Previous studies have emphasized the role of zinc-binding groups in enhancing the activity of zinc metalloprotein inhibitors^48,49, supporting the potent inhibitory potential of A. socotranus extracts. These findings suggest that the phytoconstituents identified in this study could serve as promising GLO-I inhibitors, contributing to the development of novel cancer therapeutics.

Antagonist-like inhibition of inflammatory mediators

The salivary secretions of hematophagous organisms are vital for subverting host immune and hemostatic responses, thereby ensuring successful blood feeding. These secretions effectively neutralize host-derived proinflammatory and prohemostatic mediators such as eicosanoids, histamine, serotonin, and cysteinyl leukotrienes—agents that induce localized pain, swelling, pruritus, and vasoconstriction. Additionally, platelet-derived factors like thromboxanes, serotonin, and catecholamines promote platelet aggregation and vascular constriction, further hindering feeding. To counter these challenges, blood-feeding organisms produce antagonists—salivary proteins that scavenge soluble inflammatory mediators instead of inhibiting their receptors. Known antagonist families include lipocalins, D7 proteins, and yellow proteins.

Our study introduces tablysin-15, a CAP domain protein, as a novel antagonist. Structural modeling revealed a conserved eicosanoid-binding pocket situated between helices H1, H3, and H4. Unlike other CAP proteins such as Ves v 5 or Sol i 3, tablysin-15 features a solvent-accessible hydrophobic cavity, analogous to mosquito D7 proteins, allowing efficient binding of leukotrienes LTC₄, LTD₄, and LTE₄. Ligand accommodation involves deep burial of the hydrophobic fatty acid tail within the cavity, while the polar head interacts with surface residues through hydrogen bonding or salt bridges. These structural features enable tablysin-15 to effectively bind and sequester inflammatory mediators, underscoring its functional role in immune evasion.

In search of natural antagonist-like inhibitors, we examined methanol extracts of Acridocarpus socotranus using UHPLC-MS and molecular docking. Several phytoconstituents showed promising interactions with the tablysin-15 binding pocket. Key residues involved included Val-50, Val-54, Val-126, Leu-156, Ile-43, Ile-47, Leu-106, Phe-108, His-53, His-130, Trp-59, Lys-133, Arg-40, Glu-34, and Cys-186(Xu et al., 2012). Compounds such as Cpd6 and Cpd23 interacted with His-130 and His-53, forming hydrogen bonds and water-mediated contacts (Fig. 3). Cpd18 targeted His-53, while Cpd25 showed binding with Lys-133, a site also contacted by the co-crystallized ligand. Cpd12 and Cpd13 engaged multiple residues (Arg-40, Lys-37, Glu-34) as shown in Fig. 3, and are important for ligand recognition. Moreover, Cpd19 and Cpd23 demonstrated strong interactions with Cys-186, which is involved in ligand stabilization (Fig. 3).

Fig. 3.

Interaction between ligands (compounds 5, 9, 10,12, 13, and 18) and protein (PDB ID: 3U3U).

Furthermore, Cpd9, Cpd10, and Cpd12 exhibited multi-residue interactions across the binding cavity, indicating enhanced binding stability. The reference ligand and standard inhibitor (Std2) also interacted with critical residues such as Trp-59, His-53, His-130, Lys-133, Leu-36, Glu-43, Arg-40, and Lys-37, validating the relevance of these sites as shown in Fig. 3 These results collectively suggest that A. socotranus phytochemicals can mimic natural antagonist and possess anti-inflammatory or anti-hemostatic potential.

In addition to their antagonist-like activity, molecular docking studies revealed that several of these compounds also displayed binding affinity to glyoxalase I (Glo-I), an enzyme involved in detoxifying methylglyoxal and regulating oxidative stress. Specific compounds demonstrated interactions with active site residues of Glo-I, suggesting potential inhibitory activity. This dual functionality—targeting both inflammatory mediators and oxidative stress pathways—positions these phytochemicals as promising candidates for the development of multifunctional therapeutic agents. Their ability to modulate both eicosanoid activity and glyoxalase I function expands their relevance for treating inflammatory conditions, metabolic disorders, and oxidative stress-associated diseases.

In addition, tautomeric forms of the tested compounds were also considered during the evaluation. These tautomeric variants did not produce stable or suitable docking conformations, and therefore the effective forms reported here were used for the final analysis Fig. 4.

Fig. 4.

UHPLC-MS spectra of Acridocapus socotranus methanol extract (negative ionization mode).

Conclusion

This study demonstrates that Acridocarpus socotranus is a rich source of bioactive compounds with significant potential as dual-function therapeutic agents. Phytochemical screening revealed a diverse array of metabolites—including phenolics, flavonoids, alkaloids, and terpenoids—some of which exhibited a strong affinity for the active site of glyoxalase I (Glo-I), a critical enzyme implicated in oxidative stress and cancer progression, particularly colorectal carcinoma. Molecular docking confirmed that specific phyto-constituents effectively coordinate with the catalytic zinc ion of Glo-I, mimicking the binding behavior of known synthetic inhibitors and stabilizing the enzyme-ligand complex.

In parallel, these compounds also demonstrated antagonist-like activity by interacting with the eicosanoid-binding pocket of the salivary protein tablysin-15, suggesting anti-inflammatory and anti-hemostatic properties. Key residues involved in ligand stabilization and recognition were shared between several candidate compounds and known antagonists, further validating their functional potential. The ability of these phytochemicals to bind targets relevant to both inflammation and cancer indicates a unique multi-target mechanism.

Additionally, the methanol extract of A. socotranus showed anti-proliferative effects against colorectal cancer (HCT-116 and SW480) cell lines, supporting its therapeutic relevance and aligning with the Glo-I inhibitory activity. These findings collectively position A. socotranus as a promising candidate for the development of novel anticancer and anti-inflammatory agents. Future studies focusing on bioactivity-guided fractionation, isolation, and structural optimization of lead compounds are strongly recommended to advance their potential for evaluation in vivo and in further studies, particularly in colorectal carcinoma treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Authors contributions Conceptualization: N.A.A.A.; Original draft writing: N.A.A.A., W.M.M &M.A.F Data analysis: I.A.M., N.M.M, Q.B, S.A., N.A& A.N. Investigation: I.A.M, N.M.M, S. A., N. A., R.N. B., I.A.A.A., W.M.M. & A. K., Supervision: N.A.A.A.

Data availability

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval and consent to participate

This research does not involve human participants, animals, or sensitive personal data. Therefore, ethical approval and consent to participate are not applicable.